Abstract. Histone protein lysine (Lys) methylation is an epigenetic regulator of gene expression. Histone Lys methyltransferases (HKMTs or ?writers?) install methylated Lys (KMen, n = 1-3) at specific positions, and Lys methylation recruits a diverse family of ?reader proteins? that bind these dynamic post translational modifications and induce downstream events leading to either initiation or silencing of transcription. Dysregulation in these events is associated with a wide range of diseases including cancer. While these reader and writer proteins are potential medicinal targets, few studies have probed the mechanism by which they recognize native KMen substrates or by which small molecules or histone mutations lead to their inhibition. This proposal aims to determine the balance of forces that provide binding affinity, selectivity, and catalysis for KMen as well as recently discovered inhibitors of these protein-protein interactions with the aim of gaining insights that will further the effort to develop inhibitors for these proteins. To this end, the mechanism of KMen recognition will be investigated through a combination of protein- and ligand-directed structure-activity relationships. Complementary methodology will be developed for the site-selective incorporation of electronically tuned unnatural amino acids, including substituted-phenylalanine and tyrosine residues and fluorinated aromatic residues. Using this methodology in conjunction with established techniques, the electronics of aromatic and charged residues will be systematically altered to determine the contribution of cation-pi interactions, van der Waals interactions, the hydrophobic effect, and salt bridges on affinity and selectivity across a range of di- and tri-methyl lysine reader proteins marked by subtly different binding pockets. Additionally, the role of aromatic residues in the active site of HKMTs will be investigated with respect to both catalysis and inhibition. In all cases, X-ray crystallography will be used to provide structural insights into the mechanism of recognition. Additionally, the mechanism of binding to methyl lysine mimetics and known inhibitors of reader and writer proteins will be characterized to determine whether novel mechanisms for binding and inhibition are feasible. In total, these comprehensive studies will provide a quantitative framework for the development of high quality molecular probes and next-generation inhibitors with the degree of affinity and selectivity necessary for application to disease. Furthermore, this work should readily extend to other important protein families, including methyl lysine erasers and writers as well as methyl arginine readers, writers, and erasers.